私たちは励ましのために、私たちに機器を貸し出すためのカルダン腕周囲長、キンバリージャミソン、マシューマクヘンリー、およびラジュMetherateを製造するための後半ルディリンブルフに感謝し、Almutケルバーと健太郎有川。この作品は、ADBに国立科学財団（NSF）大学院研究KJMへのフェローシップとNSF助成金IOS-1257627によってサポートされていました

著者は、彼らが競合する金融利害関係を持たないことを宣言します。

細胞内記録が原因関与する多くの技術的な手順に習得するのが困難な技術であることができます。成功した実験のために、いくつかの重要な点を考慮する必要があります。まず、実験が行われた正常振動絶縁テーブルを有することが重要です。多くの研究者が完全に優れた振動絶縁を与え、ベースから卓上を分離する空気テーブルを使用します。私たちのセットアップは、マイクロマニピュ?...

例えば、神経細胞などの細胞の電気的特性は、電圧又は電流の変化として、細胞膜を横切るイオンの流れを測定することによって観察されます。電気種々の技術は、細胞内の生体事象を測定するために開発されてきました。動物の眼で見られるニューロンはアクセス可能であり、その回路は、これらの細胞を電気生理学的研究のための良い候補を作り、多くの場合、脳内のよりも複雑ではありません。目の中の電気生理学の一般的なアプリケーションは、電図（ERG）1,2および微小電極の細胞内記録を含みます。 ERGは、動物の眼内または上に電極を配置し、光刺激を印加し、全ての周辺セル3-6の応答の和として電圧の変化を測定することを含みます。一つは、個々の光受容細胞の分光感度を特徴付けるにおいて特に興味がある場合、多くの場合、複数の細胞型は、同時に与えられた刺激とは異なる強度で応答します。したがって眼におけるスペクトル的に類似した光受容体細胞のいくつかの異なる種類がある場合は特にERGデータから特定の細胞型の感度を決定することは困難です。 1つの可能な解決法は、眼の過半数R1-6細胞で発現関心の感光体（オプシン）遺伝子を有するトランスジェニックショウジョウバエを作成し、ERGを7を実行することです。この方法の潜在的な欠点は、感光体タンパク質8、およびトランスジェニック動物の作製およびスクリーニングのための長い時間枠の低発現に何が含まれていません。スペクトル的に別個の光受容体の少ない種類の目のために、着色されたフィルタを使用して、目の適応は、それによって分光感度極大9の推定を可能にして、ERGにいくつかの細胞型の寄与を低下させることを支援することができます。 細胞内記録は、微細な電極は、セルをimpales別の技術であり、刺激が適用されます。電極レコードだけがINDIVidual細胞の応答の記録と、複数の個々の細胞を分析して、生理学的に異なる細胞型の特定の感度10-14を得ることができるようにします。我々のプロトコルは、分光感度の分析に焦点を当てているが、シャープな電極を有する細胞内記録の基本的な原理は、他​​のアプリケーションに修正可能です。例えば、試料の異なる調製物を用い、かつシャープな石英電極を用いて、1は、提起された課題に応じて、脳内の視神経葉や他の地域でより深いから記録することができます。例えば、個々の光受容体細胞15の応答時間は、視神経内の細胞活性は、19にも同様の技術を用いて記録することができ、または色刺激は、偏光20と置き換えることができる16（薄層、髄質またはlobula 17）、脳18または他の神経節のローブ-22または運動は23,24を刺激しました。 光情報伝達、光による処理エネルギーが吸収され、電気信号に変換され、ほぼすべての今日の動物門25に共通の古代の特色です。視細胞で見られ、視覚的な光伝達を開始する責任視覚色素はロドプシンです。すべての動物におけるロドプシンは、網膜または類似の分子26,27由来するオプシンタンパク質、7膜貫通Gタンパク質共役受容体ファミリーのメンバー、および関連する発色団で構成されています。オプシンのアミノ酸配列と発色団の構造は、光の異なる波長にロドプシンの吸光度に影響を与えます。光子は、発色団に吸収されるとロドプシンは、最終的には膜結合型イオンチャンネル28の開口部につながる細胞内のGタンパク質のカスケードを開始する、活性化。ほとんどのニューロンとは異なり、光受容体細胞を光刺激の変化に応答振幅の相対的な変化として測定することができる段階的な電位変化を受けます。一般的に与えられました感光体の種類が唯一のオプシン遺伝子を発現する（例外は8,10,29-31存在しますが）。洗練されたカラービジョンは、多くの脊椎動物と節足動物に見られる種類のもので、それぞれが1つまたは時折それ以上のロドプシン型を発現する光受容細胞の数百または数千の複雑な目で達成されます。視覚情報は、色や動きの完全な画像の認識結果として、眼と脳内の複雑な下流の神経シグナリングを介して感光体モザイクを超える応答を比較することによって捕捉されます。 細胞内記録を介して異なる波長の光を光受容細胞の生の応答を測定した後、その分光感度を算出することができます。この計算は、光受容体細胞の応答は、それが32を吸収した光子の特定の特性にそれが吸収する光子の数に依存するが、ではないことを述べてUnivarianceの原理に基づいています。 absoである任意の光子応答の同じ種類を誘導するロドプシンによってrbed。実際には、これは、細胞の生の応答振幅が原因（より多くの光子が吸収する）、 またはそのピーク感度（その波長を吸収するロドプシンの確率が高い）に向けて波長のシフトに光強度の増加のいずれかに増加することを意味します。我々は、既知の強度と異なる波長での応答に同じ波長と同じ強度が、未知の相対感度で細胞応答を関連で、この原理を利用しています。細胞型は、しばしば波長におけるそれらの感度ピークによって識別されます。 ここでは、より広い研究コミュニティには、この方法は、よりアクセスしやすくに焦点を当てて、細胞内記録と蝶の眼における光受容体の分光感度の分析のための1つの方法を示します。細胞内記録は、特に昆虫における色覚に関して、文献に共通のままであるが、我々はthaのを発見しました材料および方法のトンの記述は、通常、技術の再生を可能にするにはあまりにも短いです。私たちは、その簡単に複製を可能にする目的で、ビデオ形式で、この方法を提示します。我々はまた、容易に入手可能で手頃な価格の機器を用いた手法について説明します。私たちは、新しい、複雑な技術を最適化する際、研究を遅く頻繁に報告されていない一般的な注意事項に取り組みます。

<table><tbody><tr><td>Butterfly pupae</td><td></td><td></td><td>Several local species available, need USDA permits for shipping. Carolina Bio Supply has several insect species that may be ordered within the U.S. without the need for additional permits</td></tr><tr><td>Large plastic cylinder</td><td></td><td></td><td>Any chamber that remains humidified will work</td></tr><tr><td>Insect pins, size 2</td><td>BioQuip</td><td>1208B2</td><td></td></tr><tr><td>100% Desert Mesquite Honey</td><td>Trader Joe&#039;s</td><td></td><td>Any honey or sucrose solution will work</td></tr><tr><td>Xenon Arc Lamp</td><td>Oriel Instruments</td><td>66003</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Universal Power Supply</td><td>Oriel Instruments</td><td>68805</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Optical Track</td><td>Oriel Instruments</td><td>11190</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Rail Carrier, Large (2x)</td><td>Oriel Instruments</td><td>11641</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Rail Carrier, Small (4x)</td><td>Oriel Instruments</td><td>11647</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Thread Adaptor, 8-32 Male to 1/4-20 Male, pack of 10</td><td>Newport Corporation</td><td>TA-8Q20-10</td><td></td></tr><tr><td>Optical Mounting Post, 1.0 in., 0.5 in. Dia. Stainless, 8-32 &amp;amp; 1/4-20 (5x)</td><td>Newport Corporation</td><td>SP-1</td><td></td></tr><tr><td>No Slip Optical Post Holder, 2 in., 0.5 in. Diameter Posts, 1/4-20 (5x)</td><td>Newport Corporation</td><td>VPH-2</td><td></td></tr><tr><td>Fixed lens mount, 50.8 mm</td><td>Newport Corporation</td><td>LH-2</td><td></td></tr><tr><td>Fixed lens mount, 25.4 mm</td><td>Newport Corporation</td><td>LH-1</td><td></td></tr><tr><td>Condenser lens assembly</td><td>Newport Corporation</td><td>60006</td><td></td></tr><tr><td>Convex silica lens, 50.8 mm</td><td>Newport Corporation</td><td>SPX055</td><td></td></tr><tr><td>Six Position Filter Wheel, x2</td><td>Newport Corporation</td><td>FW1X6</td><td></td></tr><tr><td>Filter Wheel Mount Hub</td><td>Newport Corporation</td><td>FWM</td><td></td></tr><tr><td>Concave silica lens, 25.4 mm</td><td>Newport Corporation</td><td>SPC034</td><td></td></tr><tr><td>Collimator holder</td><td>Newport Corporation</td><td>77612</td><td></td></tr><tr><td>Collimating beam probe</td><td>Newport Corporation</td><td>77644</td><td></td></tr><tr><td>Ferrule Converter, SMA Termination to 11 mm Standard Ferrule</td><td>Newport Corporation</td><td>77670</td><td>This adapter allows the fiber optic to fit into the collimator holder&amp;nbsp;</td></tr><tr><td>600 &amp;mu;m diameter UV-vis fiber obtic cable</td><td>Oriel Instruments</td><td>78367</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Shutter with drive unit</td><td>Uniblitz</td><td>100-2B</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 0.1 OD</td><td>Newport</td><td>FRQ-ND01</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 0.3 OD</td><td>Newport</td><td>FRQ-ND03</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 0.5 OD</td><td>Newport</td><td>FRQ-ND05</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 1.0 OD</td><td>Newport</td><td>FRQ-ND10</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 2.0 OD</td><td>Newport</td><td>FRQ-ND30</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 3.0 OD</td><td>Newport</td><td>FRQ-ND50</td><td></td></tr><tr><td>LS-1-Cal lamp</td><td>Ocean Optics</td><td>LS-1-Cal</td><td></td></tr><tr><td>Spectrometer</td><td>Ocean Optics</td><td>USB-2000</td><td></td></tr><tr><td>SpectraSuite Software</td><td>Ocean Optics</td><td></td><td></td></tr><tr><td>Interference bandpass filter, 300 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67749</td><td></td></tr><tr><td>Interference bandpass filter, 310 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67752</td><td></td></tr><tr><td>Interference bandpass filter, 320 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67754</td><td></td></tr><tr><td>Interference bandpass filter, 330 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67756</td><td></td></tr><tr><td>Interference bandpass filter, 340 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65614</td><td></td></tr><tr><td>Interference bandpass filter, 350 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67757</td><td></td></tr><tr><td>Interference bandpass filter, 360 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67760</td><td></td></tr><tr><td>Interference bandpass filter, 370 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67761</td><td></td></tr><tr><td>Interference bandpass filter, 380 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67762</td><td></td></tr><tr><td>Interference bandpass filter, 390 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67763</td><td></td></tr><tr><td>Interference bandpass filter, 400 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65732</td><td></td></tr><tr><td>Interference bandpass filter, 410 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65619</td><td></td></tr><tr><td>Interference bandpass filter, 420 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65621</td><td></td></tr><tr><td>Interference bandpass filter, 430 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65622</td><td></td></tr><tr><td>Interference bandpass filter, 440 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67764</td><td></td></tr><tr><td>Interference bandpass filter, 450 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65625</td><td></td></tr><tr><td>Interference bandpass filter, 460 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67765</td><td></td></tr><tr><td>Interference bandpass filter, 470 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65629</td><td></td></tr><tr><td>Interference bandpass filter, 480 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65630</td><td></td></tr><tr><td>Interference bandpass filter, 492 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65633</td><td></td></tr><tr><td>Interference bandpass filter, 500 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65634</td><td></td></tr><tr><td>Interference bandpass filter, 510 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65637</td><td></td></tr><tr><td>Interference bandpass filter, 520 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65639</td><td></td></tr><tr><td>Interference bandpass filter, 532 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65640</td><td></td></tr><tr><td>Interference bandpass filter, 540 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65642</td><td></td></tr><tr><td>Interference bandpass filter, 550 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65644</td><td></td></tr><tr><td>Interference bandpass filter, 560 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67766</td><td></td></tr><tr><td>Interference bandpass filter, 570 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67767</td><td></td></tr><tr><td>Interference bandpass filter, 580 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65646</td><td></td></tr><tr><td>Interference bandpass filter, 589 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65647</td><td></td></tr><tr><td>Interference bandpass filter, 600 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65648</td><td></td></tr><tr><td>Interference bandpass filter, 610 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65649</td><td></td></tr><tr><td>Interference bandpass filter, 620 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65650</td><td></td></tr><tr><td>Interference bandpass filter, 632 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65651</td><td></td></tr><tr><td>Interference bandpass filter, 640 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65653</td><td></td></tr><tr><td>Interference bandpass filter, 650 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65655</td><td></td></tr><tr><td>Interference bandpass filter, 660 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67769</td><td></td></tr><tr><td>Interference bandpass filter, 671 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65657</td><td></td></tr><tr><td>Interference bandpass filter, 680 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67770</td><td></td></tr><tr><td>Interference bandpass filter, 690 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65659</td><td></td></tr><tr><td>Interference bandpass filter, 700 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67771</td><td></td></tr><tr><td>Faraday cage</td><td></td><td></td><td>Any metal structure will work that can be grounded and that fits the experimental setup.</td></tr><tr><td>Stereomicroscope, 6X, 12X, 25X, 50X magnification</td><td>Wild Heerbrugg</td><td>Wild M5</td><td>Any Stereomicroscope will do</td></tr><tr><td>Microscope stand with swinging arm and heavy base</td><td>McBain Instruments</td><td></td><td>Any heavy base with arm will do</td></tr><tr><td>Cardan arm</td><td></td><td></td><td>Custom built, See &lt;strong&gt;Figure 4&lt;/strong&gt;</td></tr><tr><td>Fiber-lite high intensity illuminator</td><td>Dolan-Jenner</td><td>MI-150</td><td>For lighting specimen</td></tr><tr><td>Fiber-lite goose-neck light guide</td><td>Dolan-Jenner</td><td>EEG 2823</td><td>Any goose-neck light guide will do</td></tr><tr><td>Marble table</td><td></td><td></td><td></td></tr><tr><td>Raised wooden table</td><td></td><td></td><td>Hole should be cut through this table so that the sandbox can rest on the marble table underneath</td></tr><tr><td>Wooden box filled with sand</td><td></td><td></td><td>custom built, any box with sand</td></tr><tr><td>Manipulator</td><td>Carl Zeiss - Jena</td><td></td><td></td></tr><tr><td>Electrode holder</td><td></td><td></td><td></td></tr><tr><td>Specimen stage</td><td></td><td></td><td></td></tr><tr><td>Alligator clip wires for grounding</td><td></td><td></td><td></td></tr><tr><td>Insulated copper wire</td><td></td><td></td><td></td></tr><tr><td>Silver wire, 0.125 mm diameter</td><td>World Precision Instruments</td><td>AGW0510</td><td></td></tr><tr><td>BNC cables</td><td></td><td></td><td></td></tr><tr><td>Preamplifier with headstage</td><td>Dagan Corporation</td><td>IX2-700</td><td></td></tr><tr><td>Humbug Noise reducer</td><td>Quest Scientific</td><td>Humbug</td><td></td></tr><tr><td>Oscilloscope, 30 MHz, 2 CH, Dual Trace, Alt-triggering, without probe</td><td>EZ Digital</td><td>os-5030</td><td></td></tr><tr><td>BNC T-adapter</td><td></td><td></td><td></td></tr><tr><td>Powerlab hardware 2/20</td><td>ADI instruments</td><td>ML820</td><td></td></tr><tr><td>Labchart software</td><td>ADI instruments</td><td>Chart 5</td><td></td></tr><tr><td>10 MHz Pulse Generator</td><td>BK Precision</td><td>4030</td><td></td></tr><tr><td>Glass pipette puller</td><td>Sutter Instruments</td><td>P-87</td><td></td></tr><tr><td>Borosillicate glass capillaries with filament</td><td>World Precision Instruments</td><td>1B120F-4</td><td></td></tr><tr><td>Potassium chloride, 3 M</td><td></td><td></td><td></td></tr><tr><td>Slotted plastic tube</td><td></td><td></td><td></td></tr><tr><td>Low melting temperature wax</td><td></td><td></td><td></td></tr><tr><td>Soldering Iron</td><td>Weller</td><td></td><td></td></tr><tr><td>Platform with ball-and-socket magnetic base</td><td>Hama photo and video</td><td></td><td></td></tr><tr><td>Double edge carbon steel, breakable razor blade</td><td>Electron Microscopy Sciences</td><td>72004</td><td></td></tr><tr><td>Vaseline</td><td></td><td></td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td></td><td></td></tr></tbody></table>

determination of photoreceptor cell spectral sensitivity in an insect model from in vivo intracellular recordings

細胞内記録は、単一の細胞が与えられた刺激に応答することができる方法を決定するために使用される強力な手法です。ビジョン研究では、細胞内記録は、歴史的に今日でも使用されている異なる光刺激に対する個々の光受容細胞の感度を研究するために使用される一般的な技術となっています。しかし、目に細胞内記録実験を複製することを望む研究者のための文献で詳細な方法論の不足が残っています。ここでは、より一般的には目の生理機能を調べるためのモデルとして昆虫を提示します。昆虫視細胞は、眼の表面付近に位置するので、簡単に行くことができている、とビジョンに関与する機構の多くは、動物門を越えて保存されています。私たちは、電子が少し前に経験を持つ研究者へのこの技術は、よりアクセスしやすくすることを目標に、蝶の眼における光受容体細胞のin vivoでの細胞内記録のための基本的な手順を説明しますlectrophysiology。我々は、単一の細胞内へのガラス微小電極、最後に記録手順自体を挿入する方法、記録のためのライブ蝶を準備する方法、必要な基本的な設備を導入します。我々はまた、個々の細胞型の分光感度を測定するための生のレスポンスデータの基本的な分析を説明します。我々のプロトコルは、分光感度を決定することに焦点を当てているが、他の刺激（ 例えば 、偏光）及び方法のバリエーションは、このセットアップに適用可能です。

録画設定の多くの要素については、書面による説明は十分な詳細情報を提供していません。 図1は、完全な記録のセットアップに含まれるコンポーネントの概略図です。 図2において、スペクトルは補正係数が必要とされる理由の感覚、何この補正を計算するために必要とさを与えるために、白色光、各干渉フィルタのためにプロットされている。...

Watch this Scientific Journal Video about Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings at JoVE.com

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

細胞内記録の電気生理学的技術を実証し、バタフライの複眼における単一の光受容体細胞の分光感度を決定するために使用されます。

昆虫モデルにおける視細胞分光感度の決意から<em&gt;インビボ</em&gt;細胞内記録

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...

determination-photoreceptor-cell-spectral-sensitivity-an-insect-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

11.3K ビュー.  University of California, Irvine. 細胞内記録の電気生理学的技術を実証し、バタフライの複眼における単一の光受容体細胞の分光感度を決定するために使用されます。 

ビデオ: Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

胚の脊髄から交連ニューロンの解剖と文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

神経科学

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

シングルユニット in vivoで</emラットの視交叉から>録音

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

無傷の網膜におけるGFP発現細胞集団の電気生理学的特性

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行性蛍光標識は、特定された神経細胞から標的細胞外シングルユニット記録を可能にします<em生体内で&gt;</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

の記録細胞内の電圧応答のための電気生理学的手法<em&gt;ショウジョウバエ</em&gt;光刺激に感光体と介在ニューロン<em&gt;インビボ</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

全細胞電圧クランプ記録のための電気生理学的方法<em&gt;ショウジョウバエ</em&gt;感光体

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor cells in isolated eyestalks using the discontinuous single electrode-switched voltage-clamp configuration. First, with a razor blade we made an opening in the dorsal cornea to get access to the retina. Thereafter, we inserted a glass electrode through the opening, and penetrated a cell as reported by the recording of a negative potential. Membrane potential was clamped at the photoreceptor's resting potential and a light-pulse was applied to activate currents. Finally, the two light-flash protocol was employed to measure current desensitization and recovery. The first light-flash triggers, after a lag period, the transduction ionic current, which after reaching a peak amplitude decays towards a desensitized state; the second flash, applied at varying time intervals, assesses the state of the light-activated conductance. To characterize the light-elicited current, three parameters were measured: 1) latency (the time elapsed between light flash delivery and the moment in which current achieves 10% of its maximum value); 2) peak current; and 3) desensitization time constant (exponential time constant of the current decay phase). All parameters are affected by the first pulse.
To quantify recovery from desensitization, the ratio p2/p1 was employed versus time between pulses. p1 is the peak current evoked by the first light-pulse, and p2 is the peak current evoked by the second pulse. These data were fitted to a sum of exponential functions. Finally, these measurements were carried out as function of circadian time.

A protocol for the study of desensitization and sensitivity recovery of crayfish photoreceptors as a function of circadian time is presented.

光刺激の送達時のザリガニ光受容体の脱感作と回復

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor ...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

ショウジョウバエ光受容体の光色素レベルを測定するための電気生理学的方法

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...

In Vivo Intracellular Recording of Photoreceptor Cell Spectral Sensitivity in a Butterfly

Source: McCulloch, K. J., et al.. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53829">Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings.</a> J. Vis. Exp. (2016)
This video demonstrates the procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly and determining the spectral sensitivity of individual cell types. This technique enables the study of sensitivities of individual photoreceptor cells to different light stimuli.

蝶の光受容体細胞スペクトル感度のin vivo細胞内記録

1. Specimen Prep and Recording Procedure

	Prepare the Specimen
	
		Affix an individual butterfly inside a small plastic tube with hot wax so the head is ...

JoVE Encyclopedia of Experiments

Neurophysiology

Imaging and Optical Techniques

Electrophysiological Recording of Voltage Responses of Drosophila Retinal Photoreceptors to Light Stimuli

Source: Juusola, M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54142">Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo.</a> J. Vis. Exp. (2016).
This video outlines the protocol for electrophysiological recording of photoreceptor activity in the eye of an immobilized Drosophila, enabling precise measurement of voltage changes in response to light stimuli.

光刺激に対するショウジョウバエ網膜光受容体の電圧応答の電気生理学的記録

1. Recording from R1-R6 Photoreceptors 

	Always be grounded when operating the microelectrode amplifier (for example by touching the metal surface of the ...

昆虫モデルにおける視細胞分光感度の決意から

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胚の脊髄から交連ニューロンの解剖と文化

シングルユニット in vivoで録音

無傷の網膜におけるGFP発現細胞集団の電気生理学的特性

逆行性蛍光標識は、特定された神経細胞から標的細胞外シングルユニット記録を可能にします

の記録細胞内の電圧応答のための電気生理学的手法

全細胞電圧クランプ記録のための電気生理学的方法

光刺激の送達時のザリガニ光受容体の脱感作と回復

ショウジョウバエ光受容体の光色素レベルを測定するための電気生理学的方法

蝶の光受容体細胞スペクトル感度のin vivo細胞内記録

光刺激に対するショウジョウバエ網膜光受容体の電圧応答の電気生理学的記録